Multiplexed analysis of nucleic acid hybridization thermodynamics using integrated arrays

ABSTRACT

The present disclosure provides methods and devices for simultaneous identification of a plurality of target nucleic acid sequences in a single sample chamber that includes an addressable array of nucleic acid probes attached to a solid surface. Addressable signals can be generated and measured, in real-time, upon hybridization of target sequences at the individual probe locations within the array while the temperature of the system is varied. Such generated signals, as a function temperature, can then be used to compute the properties of nucleic acid hybridization at each addressable location which is ultimately utilized to estimate the sequence of the target nucleic acids. In particular, an integrated semiconductor biosensor array device can be used to measure the addressable signals.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 9, 2015, isnamed 42500-716.201 SL.txt and is 3,603 bytes in size.

BACKGROUND

DNA-DNA hybridization is a molecular biology technique that measures thedegree of sequence similarity between deoxyribonucleic acid (DNA)polymers (polynucleotides). The underlying principle is that thebuilding blocks of the DNA polymer, i.e., nucleotides, include specificnitrogen-containing nucleobases (guanine “G,” adenine “A,” thymine “T,”and cytosine “C”) capable of pairing up with complementary nucleobases(A with T and C with G) to form hydrogen bonds (two (2) between A-T andthree (3) between C-G). Therefore, DNA moieties with complementarysequences have an affinity to bind (hybridize) to one another and DNAdimers (double stranded DNA structures). The thermodynamicscharacteristics hybridization depends predominately on the total number,and strength of hydrogen bonds formed between the DNA moieties; aquantity which is a function of multiple parameters such ascomplementary nucleobase (base) stretches, non-complementary gaps, andthe concentration and variety of anions and cations in the environment.

The thermodynamic characteristic of DNA-DNA-hybridization is a powerfultool to infer sequence information regarding the participating moieties.The “gold standard” method to extract such information is melt curveanalysis (MCA) which detects the dissociation-characteristics ofdouble-stranded and hybridized DNA dimers during a gradual heatingprocess. As temperature is raised, the DNA-DNA complex (assembledthrough multiple hydrogen bonds) becomes less stable and the strandsbegin to dissociate. Thus, by monitoring the concentration of hybridizedcomplexes versus temperature, one can evaluate the stability of thecomplex as a function of temperature and correlate it to alterationswithin the target sequence (and hydrogen bonds) and, for example,identify single-nucleotide polymorphisms (“SNPs”) orinsertions/deletions (“indels”).

SUMMARY

Recognized herein are various limitations associated with current andprevious MCA techniques. Originally, MCA was enabled using UV absorbancemeasurements (Ansevin, et al., Biopolymers, 1976); however techniquesbased on fluorescence measurements using, for example, deoxyribonucleicacid (DNA)-intercalating fluorophores such as SYBR Green are more commontoday (Wittwer C. T., et al., BioTechniques, 22:130-138, 1997, Ririe K.M., et al., Anal. Biochem, 245:154-160, 1997, Lipsky, R. H., et al.,Clin. Chem. 47:635-644, 2001, and Wittwer C. T., et al. U.S. Pat. No.7,785,776).

While MCA-based methods offer approaches to measure hybridizationthermodynamics, they have very limited multiplexing capabilities, i.e.,analyzing multiple simultaneously occurring DNA hybridization reactionsin a single reaction chamber. In the case of intercalating dyes forexample, the measured fluorescent signal is basically the aggregate ofall signals originating from individual hybridization events in thereaction chamber, and therefore difficult to decipher when DNA moietieshave similar thermodynamic characteristic, or when one moiety has asignificantly larger concentration and signal compared to others.

Detecting the level of DNA hybridization at a constant temperature alsohas use in identifying specific sequences. DNA microarray platforms,sometimes referred to as genechips, typically operate based on thisprincipal (Schena M., Shalon D., Davis R. W., Brown P. O. “Quantitativemonitoring of gene expression patterns with a complementary DNAmicroarray,” Science 270 1995(5235): 467-470, and Stoughton RB,“Applications of DNA microarrays in biology,” Annu Rev Biochem. 2005;74:53-82). The advantage of microarrays is that they employ the DNAhybridization thermodynamics to identify sequence, in a massivelyparallel fashion. However, they lack the specificity of MCA methods.This is a fundamental limitation and is rooted in the fact thatmicroarrays can measure hybridization at a single temperature point,i.e., the temperature in which the sample in incubated on the array fora fixed duration of time, before washing and imaging. While the datafrom microarrays is still useful to identify SNPs or indels, it is notthorough in terms of thermodynamics. Generally speaking, it is alsodifficult to design a large number of immobilized probes in for amicroarray that can discriminate properly between nucleic acid targetsat a single temperature point. This problem becomes particularlychallenging when the CG content of the targets has a large variation(>20%) or when targets include highly stable hairpin monomer structure.

The present disclosure provides methods, devices and systems to measurethe thermodynamic characteristics of multiple nucleic acid hybridizationreactions that concurrently happen in real time in a single reactionchamber. Various embodiments provided herein can be used to createunique nucleic acid detection platform for applications such moleculardiagnostics, nucleic acid (e.g., DNA) forensics, and pathogengenotyping, to name a few. Further embodiments are provided which cantake advantage of semiconductor-integrated biosensor arrays to bothminiaturize and integrate the required detection devices.

An aspect of the present disclosure provides a method for assaying apresence of a target nucleic acid molecule in a sample, comprising (a)providing a chip comprising an integrated sensor adjacent to a samplechamber, wherein the sample chamber is configured to retain the samplehaving or suspected of having the target nucleic acid molecule, andwherein the integrated sensor (i) has a surface including a probe thatselectively couples to the target nucleic molecule, and (ii) detects atleast one signal from the sample, which at least one signal isindicative of a presence or absence of the target nucleic acid molecule;(b) providing the sample in the sample chamber under conditions thatpermit the probe to selectively couple to the target nucleic acidmolecule; (c) subjecting the surface to a temperature change while thesample is in the sample chamber; (d) measuring the at least one signalin real-time while subjecting the surface to the temperature change; and(e) generating signal versus temperature data using measurements of theat least one signal with the temperature change.

In some embodiments of aspects provided herein, the at least one signalincludes a plurality of signals. The plurality of signals can be atmultiple time points and/or multiple temperatures. For example,temperature can be increased at a rate that is a linear or non-linearfunction of time, and signals can be measured. In some embodiments ofaspects provided herein, the signal versus temperature data is part of amelt curve.

In some embodiments of aspects provided herein, the probe is anoligonucleotide. In some embodiments of aspects provided herein, thesample is provided in the sample chamber under conditions that permitthe oligonucleotide to hybridize to the target nucleic acid molecule. Insome embodiments of aspects provided herein, a sequence of the targetnucleic acid molecule forms a hairpin loop structure when hybridized tothe oligonucleotide. In some embodiments of aspects provided herein, theintegrated sensor is in an array of a plurality of integrated sensors inthe chip. In some embodiments of aspects provided herein, the arraycomprises at least about 100 integrated sensors, at least about 500integrated sensors, at least about 1000 integrated sensors, at leastabout 2000 integrated sensors, at least about 5000 integrated sensors orat least about 10,000 integrated sensors. In some embodiments of aspectsprovided herein, the at least one signal is selected from the groupconsisting of an optical signal, electrochemical signal andelectrostatic signal. In some embodiments of aspects provided herein,the at least one signal is an optical signal that is indicative of aninteraction between an energy acceptor and an energy donor pair. In someembodiments of aspects provided herein, the energy acceptor quenchesoptical activity of the energy donor. In some embodiments of aspectsprovided herein, the energy acceptor is coupled to one or morenucleotides of the target nucleic acid molecule. In some embodiments ofaspects provided herein, the energy acceptor is a quencher. In someembodiments of aspects provided herein, the energy donor is coupled tothe probe. In some embodiments of aspects provided herein, the energydonor is a fluorophore. In some embodiments of aspects provided herein,the interaction is not Forster resonance energy transfer (FRET). In someembodiments of aspects provided herein, the at least one signal is anoptical signal indicative of the activity of an optically-activespecies. In some embodiments of aspects provided herein, theoptically-active species is an intercalator. In some embodiments ofaspects provided herein, the optically-active species is a fluorophore.In some embodiments of aspects provided herein, the detecting comprisesmeasuring an increase in the at least one signal relative to background.In some embodiments of aspects provided herein, the detecting comprisesmeasuring a decrease in the at least one signal relative to background.In some embodiments of aspects provided herein, the integrated sensorfurther comprises an optical detector, and, in (d), the least one signalis measured with the optical detector. In some embodiments of aspectsprovided herein, the optical detector comprises a complementarymetal-oxide semiconductor (CMOS) integrated circuit (IC) device. In someembodiments of aspects provided herein, the method further comprises,prior to (a), (i) providing a reaction mixture including a biologicalsample having or suspected of having a template nucleic acid molecule asa precursor of the target nucleic acid molecule, at least one primerthat is complementary to the template nucleic acid molecule, and apolymerase, and (ii) subjecting the reaction mixture to a nucleic acidamplification reaction under conditions that yield the target nucleicacid molecule in the sample. In some embodiments of aspects providedherein, the at least one primer has a sequence that is selected toidentify single nucleotide polymorphism (SNP) in a sequence of thetarget nucleic acid molecule. In some embodiments of aspects providedherein, the nucleic acid amplification is polymerase chain reaction(PCR). In some embodiments of aspects provided herein, the nucleic acidamplification is asymmetric nucleic acid amplification. In someembodiments of aspects provided herein, the chip is electrically coupledto a computer processor that electrically receives the at least onesignal from the integrated sensor and determines the presence or absenceof the target nucleic acid molecule from the at least one signal. Insome embodiments of aspects provided herein, the computer processorgenerates the signal versus temperature data. In some embodiments ofaspects provided herein, the method further comprises outputting thesignal versus temperature data on an electronic report. In someembodiments of aspects provided herein, the electronic report isoutputted on a user interface of an electronic device of a user. In someembodiments of aspects provided herein, in (c), the surface is subjectedto the temperature change at an average rate from about 1° C./min to 20°C./min. In some embodiments of aspects provided herein, in (c), atemperature controller in thermal communication with the surfacesubjects the surface to the temperature change. In some embodiments ofaspects provided herein, the probe is coupled to the surface via alinker. In some embodiments of aspects provided herein, the linkercomprises a species selected from the group consisting of an amino acid,a polypeptide, a nucleotide and an oligonucleotide. In some embodimentsof aspects provided herein, when the at least one signal is indicativeof the presence of the target nucleic acid molecule, the target nucleicacid molecule is detected as a sensitivity of at least about 90%, atleast about 95%, at least about 98%, at least about 99%, at least about99.9% or at least about 99.99%. In some embodiments of aspects providedherein, the method further comprises determining a single nucleotidepolymorphism (SNP) in a sequence of the target nucleic acid moleculeusing the signal versus temperature data. In some embodiments of aspectsprovided herein, the method further comprises measuring at least onecontrol signal or a plurality of control signals from an additionalintegrated sensor. In some embodiments of aspects provided herein, thesignal versus temperature data is normalized against measurement(s) ofthe at least one control signal. In some embodiments of aspects providedherein, the temperature change is at a linear rate. In some embodimentsof aspects provided herein, the temperature change is from a firsttemperature to a second temperature that is greater than the firsttemperature. In some embodiments of aspects provided herein, the atleast one signal includes a plurality of signals.

Another aspect of the present disclosure provides a method for assayinga presence of a target nucleic acid molecule in a sample, comprising (a)subjecting a hybridization array having at least one integrated sensorto a temperature change, (b) measuring signals from the hybridizationarray with the at least one integrated sensor, and (c) assaying apresence of the target nucleic acid at a sensitivity of at least about90% by assessing dissociation-characteristics of the target nucleic acidmolecule with the temperature change. In some embodiments of aspectsprovided herein, the sensitivity is at least about 95%. In someembodiments of aspects provided herein, the hybridization array has aplurality of integrated sensors. In some embodiments of aspects providedherein, the at least one integrated sensor is an optical sensor.

Another aspect of the present disclosure provides a system for assayinga presence of a target nucleic acid molecule in a sample, comprising: achip comprising an integrated sensor adjacent to a sample chamber,wherein the sample chamber is configured to retain the sample having orsuspected of having the target nucleic acid molecule, and wherein theintegrated sensor (i) has a surface including a probe that selectivelycouples to the target nucleic molecule, and (ii) detects at least onesignal from the sample, which at least one signal is indicative of apresence or absence of the target nucleic acid molecule; a computerprocessor coupled to the chip and programmed to (i) subject the surfaceto a temperature change while the sample is in the sample chamber; (ii)measure the at least one signal while subjecting the surface to thetemperature change; and (iii) generate signal versus temperature datausing measurements of the at least one signal with the temperaturechange.

In some embodiments of aspects provided herein, the at least one signalincludes a plurality of signals. The plurality of signals can be atmultiple time points and/or multiple temperatures. For example,temperature can be increased at a rate that is a linear or non-linearfunction of time, and signals can be measured. In some embodiments ofaspects provided herein, the signal versus temperature data is part of amelt curve.

In some embodiments of aspects provided herein, the integrated sensor isin an array of a plurality of integrated sensors in the chip. In someembodiments of aspects provided herein, the array comprises at leastabout 100 integrated sensors, at least about 500 integrated sensors, atleast about 1000 integrated sensors, at least about 2000 integratedsensors, at least about 5000 integrated sensors or at least about 10,000integrated sensors. In some embodiments of aspects provided herein, anindividual integrated sensor of the array is individually addressable.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “FIG” and “FIGs” herein), of which:

FIG. 1 shows an exemplary schematic of a multiplex analysis system;

FIG. 2 shows an exemplary schematic of probe and target interaction withenergy donors and energy acceptors;

FIG. 3 shows an exemplary schematic of probe and target interaction withintercalators;

FIG. 4 shows an exemplary schematic of probe and target interaction withlabeled target;

FIG. 5 shows an exemplary schematic of melt curve analysis;

FIG. 6 shows exemplary images and a schematic of a biosensor array;

FIG. 7 shows an exemplary schematic of biochip array circuitry;

FIG. 8A shows exemplary images of a biochip array;

FIG. 8B shows exemplary graphs of temperature control and melt curveanalysis;

FIG. 8C shows exemplary probe and target sequences (SEQ ID NOS 1-4,respectively, in order of appearance);

FIG. 9 shows an exemplary graph of melt curve analysis and exemplaryprobe and target sequences (SEQ ID NOS 5-8, respectively, in order ofappearance);

FIG. 10A shows an exemplary graph of melt curve analysis;

FIG. 10B shows exemplary fluorophore-quencher target and probe sequences(SEQ ID NOS 3-4, 9-10, 1-2, and 11-12, respectively, in order ofappearance); and

FIG. 11 shows an exemplary schematic of a computer control system thatis programmed or otherwise configured to implement methods providedherein.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

The term “probe” as used herein generally refers to a molecular speciesor other marker that can bind to a specific target nucleic acidsequence. A probe can be any type of molecule or particle. Probes cancomprise molecules and can be bound to the substrate or other solidsurface, directly or via a linker molecule.

The term “detector” as used herein generally refers to a device,generally including optical and/or electronic components that can detectsignals.

The term “mutation” as used herein generally refers to genetic mutationsor sequence variations such as a point mutation, a single nucleotidepolymorphism (SNP), an insertion, a deletion, a substitution, atransposition, a translocation, a copy number variation, or anothergenetic mutation, alteration or sequence variation.

The term “about” or “nearly” as used herein generally refers to within+/−15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the designatedamount.

The term “label” as used herein refers to a specific molecular structurethat can be attached to a target molecule, to make the target moleculedistinguishable and traceable by providing a unique characteristic notintrinsic to the target molecule.

The present disclosure provides methods, devices, and systems to enablemultiplex detection of nucleic acid hybridization reactions, in realtime, and as a function temperature. The methods, device, and systems ofthe present disclosure can comprise components including, but notlimited to:

1. Sample chamber, which can include an aqueous environment in which aplurality of free-moving nucleic acid targets, to be analyzed, arepresent;

2. Probe array, which can comprise a plurality of nucleic acid probes atindependently (or individually) addressable locations on a solidsurface. The probe array can be interfaced with the sample chamber. Eachaddressable location (herein referred to as a “pixel”) can comprise aplurality of identical nucleic acid sequences (herein referred to as“probes”) that can specifically hybridize to a specific target;

3. Temperature controller, which can measure and adjust the temperatureof the sample chambers to predetermined or specific values between; and

4. Detector, which can measure, in parallel, the signals generated atevery pixel. Signals can be related to the molecular labels' presenceand activity in their vicinity, as the hybridization events progress asa function of temperature. The signals can be discrete (e.g.,individually resolvable) signals.

The probe array can include independently addressable locations thateach has one or a plurality of probes. Probes at a given independentlyaddressable location of the array can be different than probes at otherindependently addressable locations of the array. In some cases, probesof a group of locations of the array are the same. Probes of the groupof locations can be different than probes of all other locations of thearray.

Methods, devices, and systems of the present disclosure can employvariants of the above components assembled together to create a systemcapable of measuring nucleic acid hybridization reactions in parallel.FIG. 1 shows an example of a multiplex analysis system. The nucleicacids are in the sample chamber (or reaction chamber), where they canmove through diffusion and drift processes to interact with, and ifthermodynamically favorable hybridize to, the probes at individualpixels of the addressable array. The temperature controller can set thetemperature of the reaction chamber to various predefined values tocreate dissimilar and/or time-varying conditions for the hybridizationevents. Meanwhile, the detector can measure the quantity (or magnitude)of hybridization incidents at every pixel, in real time, and as thetemperature is varying. The acquired data are subsequently used toassess the thermodynamic characteristics of the interaction between theprobe nucleic acids and the target nucleic acids.

Reaction Chambers

Reaction chambers can comprise a closed reservoir. The reaction chambercan have a volume from about 10 nanoliters (nL) to 10 milliliters (mL).In some cases, the reaction chamber volume is from about 1 microliter(μL) to 100 μL. The reaction chamber volume can be at least about 10 nL,100 nL, 1 μL, 10 μL, 100 μL, 1 mL, or 10 mL.

Reaction chambers can contain an aqueous solution. The aqueous solutionwithin the reaction chamber can comprise a buffered saline-basedsolution, such as an aqueous solution comprising a mixture of a weakacid and its conjugate base, or vice versa. The solution can comprise aplurality of target nucleic acid sequences, herein referred to as“targets.” The term “nucleic acid sequence” or “nucleotide sequence” asused in this context refers to nucleic acid molecules with a givensequence of nucleotides, of which it is desired to know the presence oramount. The nucleotide sequence can comprise ribonucleic acid (RNA) orDNA, or a sequence derived from RNA or DNA. Examples of nucleotidesequences are sequences corresponding to natural or synthetic RNA or DNAincluding genomic DNA and messenger RNA. The length of the sequence canbe any length that can be amplified into nucleic acid amplificationproducts, or amplicons, for example up to about 20, 50, 100, 200, 300,400, 500, 600, 700, 800, 1,000, 1,200, 1,500, 2,000, 5,000, 10,000 ormore than 10,000 nucleotides in length.

In some cases, the targets can include reporter molecules, hereinreferred to as “labels.” Labels can comprise molecular structures that,once attached to a nucleic acid sequence, provide a distinctcharacteristic that is not inherent to those nucleic acid molecules.Examples are labels that create unique optical characteristics.

In some examples, optical labels are used. An optical label can be usedas single signal generating entity or part of a dual-molecule reporterin the role of either an energy donor, or energy acceptor.

Acceptors and donors can both be fluorophores molecules. Whether afluorophore is a donor or an acceptor may be based on its excitation andemission spectra, and the fluorophore with which it is paired.

Examples of energy donor/energy acceptor fluorophore pairs include, butare not limited to, cyan fluorescent protein (CFP) and yellowfluorescent protein (YFP); Cy3 and Cy5; fluorescein andtetramethylrhodamine; IAEDANS and fluorescein; EDANS and dabcyl;fluorescein and QSY 7 or QSY 9 dyes; Alexa Fluor 350 and Alexa Fluor488; Alexa Fluor 488 and Alexa Fluor 546, 555, 568, 594, or 647; AlexaFluor 546 and Alexa Fluor 568, 594, or 647; Alexa Fluor 555 and AlexaFluor 594 or 647; Alexa Fluor 568 and Alexa Fluor 647; and Alexa Fluor594 and Alexa Fluor 85.

Quenchers molecules can be used with method of the present disclosure asacceptors of a dual reporter structure. Example quenchers, withoutlimitation, include Black Hole Quencher Dyes (Biosearch Technologiessuch as BHQ-0, BHQ-1, BHQ-2, BHQ-3, BHQ-10; QSY Dye fluorescentquenchers (from Molecular Probes/Invitrogen) such as QSY7, QSY9, QSY21,QSY35, and other quenchers such as Dabcyl and Dabsyl; Cy5Q and Cy7Q andDark Cyanine dyes (GE Healthcare). Examples of fluorophore donormolecules that can be used in conjunction with above quenchers include,without limitation, fluors such as Cy3B, Cy3, or Cy5; DY-Quenchers(Dyomics), such as DYQ-660 and DYQ-661; and ATTO fluorescent quenchers(ATTO-TEC GmbH), such as ATTO 540Q, 580Q, 612Q. Quenchers can beacceptors.

Optical labels can also be nucleic acid intercalators dyes, hereinreferred to as intercalators. Examples include, but are not limited to,ethidium bromide, YOYO-1, SYBR Green, and EvaGreen. The near-fieldinteractions between energy donors and energy acceptors, betweenintercalators and energy donors, or between intercalators and energyacceptors can result in the generation of unique signals or a change inthe signal amplitude. For instance, such interactions can result inquenching (i.e., energy transfer from donor to acceptor that results innon-radiative energy decay) or Forster resonance energy transfer (FRET)(i.e., energy transfer from the donor to an acceptor that results inradiative energy decay).

Other examples of labels include electrochemical labels, electrostaticlabels, colorimetric labels and mass tags. Such labels may be used withdevices, methods and systems of the present disclosure.

Labels can be coupled to a target molecule by direct attachment or byattachment through one or more linkers (e.g., linker molecules). In somecases, labels couple to a target molecule by an electrostaticinteraction that may not involve forming a covalent bond with the targetmolecule.

The labeling of the target molecules (targets) can be performed using avariety of methods. In some examples, the labels are chemically attachedduring in-vitro amplification (e.g., by PCR) of nucleic targets usinglabelled primers. Amplification can comprise a number of differentmolecular replication or amplification approaches, including but notlimited to polymerase chain reaction (PCR), asymmetric PCR, multiplexPCR, nested PCR, hot-start PCR, touchdown PCR, RT-PCR, andmethylation-specific PCR. Amplification can be isothermal, withchemistries including but not limited to loop-mediated isothermalamplification (LAMP), strand displacement amplification (SDA),helicase-dependent amplification (HDA), and nicking enzyme amplificationreaction (NEAR). During the amplification, a labeled primer is elongatedto become an amplicon, resulting in the generated amplicon, i.e., thetarget, being labelled. Methods of attaching and/or conjugating suchlabels include, without limitation, ligation, biotin-streptavidinconjugation, hydrazone bonds, reaction of amine-reactive labels withaminoallyl dUTP, and T4 polynucleotide kinase (PNK). In other examples,the labels are attached to modified deoxyribonucleotide triphosphates(dNTPs) that are used to generate the amplicons during the amplificationprocesses. In such methods, a portion of one or more types of the dNTPsare chemically modified to have a label attach to them or to comprise achemical binding site to which a label can attach after the dNTP isincorporated into the elongated nucleic acid strand. In some cases, thelabel is a single strand DNA (ssDNA) or double strand DNA (dsDNA)binding molecule.

In some cases, amplification can be performed by PCR. PCR can rely onthermal cycling, including one or more cycles of repeated heating andcooling of the reaction for polynucleotide melting and enzymaticreplication of the polynucleotide. Primers (short nucleic acidfragments) containing sequences complementary to a target region of atarget polynucleotide along with polymerizing enzyme (e.g., DNA or RNApolymerase), can provide for the selective and repeated amplification ofthe target polynucleotide. The primers can have sequences that arecomplementary to a sequence of interest, such as a sequence with amutation or a sequence that has been identified to predispose a subjectto a given disease (e.g., cancer). As PCR progresses, the polynucleotidegenerated can itself used as a template for replication, setting inmotion a chain reaction in which the target polynucleotide template isexponentially amplified.

As an alternative, amplification can be asymmetric PCR, which canpreferentially amplify one polynucleotide strand in a double-strandedpolynucleotide template. This approach can be where amplification ofonly one of two complementary strands is required. In asymmetric PCR,PCR is carried out as described above, but with an excess of a primerhaving sequence complementarity to the strand targeted foramplification. Because of the slow (arithmetic) amplification later inthe reaction after the limiting primer has been exhausted, extra cyclesof PCR may be required. In some cases, asymmetric amplification may usea limiting primer with a higher melting temperature (Tm) than an excessprimer to maintain reaction efficiency as the limiting primerconcentration decreases mid-reaction.

Amplification can be isothermal amplification. An example of anisothermal amplification method is strand displacement amplification,also referred to as SDA, which may use cycles of annealing pairs ofprimer sequences to opposite strands of a target sequence, primerextension in the presence of a dNTP to produce a duplexhemiphosphorothioated primer extension product, endonuclease-mediatednicking of a hemimodified restriction endonuclease recognition site, andpolymerase-mediated primer extension from the 3′ end of the nick todisplace an existing strand and produce a strand for the next round ofprimer annealing, nicking and strand displacement, resulting ingeometric amplification of product. See, e.g., U.S. Pat. No. 5,270,184and U.S. Pat. No. 5,455,166, each of which is entirely incorporatedherein by reference. Thermophilic SDA (tSDA) may use thermophilicendonucleases and polymerases at higher temperatures in essentially thesame method. See, e.g., European Pat. No. 0 684 315, which is entirelyincorporated herein by reference.

Examples of other amplification methods include rolling circleamplification (RCA) (e.g., Lizardi, “Rolling Circle Replication ReporterSystems,” U.S. Pat. No. 5,854,033); helicase dependent amplification(HDA) (e.g., Kong et al., “Helicase Dependent Amplification NucleicAcids,” U.S. Pat. Appln. Pub. No. US 2004-0058378 A1); and loop-mediatedisothermal amplification (LAMP) (e.g., Notomi et al., “Process forSynthesizing Nucleic Acid,” U.S. Pat. No. 6,410,278), each of which isentirely incorporated herein by reference. In some cases, isothermalamplification utilizes transcription by an RNA polymerase from apromoter sequence, such as may be incorporated into an oligonucleotideprimer. Transcription-based amplification methods may include nucleicacid sequence based amplification, also referred to as NASBA (e.g., U.S.Pat. No. 5,130,238); methods which rely on the use of an RNA replicaseto amplify the probe molecule itself, commonly referred to as Qβreplicase (e.g., Lizardi, P. et al. (1988) Bio Technol. 6, 1197-1202);self-sustained sequence replication (e.g., Guatelli, J. et al. (1990)Proc. Natl. Acad. Sci. USA 87, 1874-1878; Landgren (1993) Trends inGenetics 9, 199-202; and Lee, H. H. et al., Nucleic Acid AmplificationTechnologies (1997)); and methods for generating additionaltranscription templates (e.g., U.S. Pat. No. 5,480,784 and U.S. Pat. No.5,399,491), each of which is entirely incorporated herein by reference.Other methods of isothermal nucleic acid amplification include the useof primers containing non-canonical nucleotides (e.g., uracil or RNAnucleotides) in combination with an enzyme that cleaves nucleic acids atthe non-canonical nucleotides (e.g., DNA glycosylase or RNaseH) toexpose binding sites for additional primers (e.g., U.S. Pat. No.6,251,639, U.S. Pat. No. 6,946,251, and U.S. Pat. No. 7,824,890), whichare hereby incorporated by reference in their entirety. Isothermalamplification processes can be linear or exponential.

Probe Arrays

A probe can comprise biological materials deposited so as to createspotted arrays. A probe can comprise materials synthesized, deposited,or positioned to form arrays according to other technologies. Thus,microarrays formed in accordance with any of these technologies may bereferred to generally and collectively hereafter for convenience as“probe arrays.” The term “probe” is not limited to probes immobilized inarray format. Rather, the functions and methods described herein canalso be employed with respect to other parallel assay devices. Forexample, these functions and methods may be applied with respect toprobe-set identifiers that can identify probes immobilized on or inbeads, optical fibers, or other substrates or media. The construction ofvarious probe arrays is described in more detail herein.

In some cases, the probe comprises a polynucleotide. The terms“polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acidmolecule” as used herein can include a polymeric form of nucleotides ofany length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA).This term refers only to the primary structure of the molecule. Thus,the term can include triple-, double- and single-stranded DNA, as wellas triple-, double- and single-stranded RNA. It can also includemodifications, such as by methylation and/or by capping, as well asunmodified forms of polynucleotides. Further, the terms“polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acidmolecule” can include polydeoxyribonucleotides (containing2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any othertype of polynucleotide which is an N- or C-glycoside of a purine orpyrimidine base, and other polymers containing nonnucleotidic backbones.Nucleic acids can comprise phosphodiester bonds (i.e., natural nucleicacids), Nucleic acids can comprise nucleic acid analogs that may havealternate backbones, comprising, for example, phosphoramide (see, e.g.,Beaucage et al., Tetrahedron 49(10):1925 (1993) and U.S. Pat. No.5,644,048), phosphorodithioate (see, e.g., Briu et al., J. Am. Chem.Soc. 11 1:2321 (1989), O-methylphosphoroamidite linkages (see, e.g.,Eckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid (PNA) backbones and linkages(see, e.g., Carlsson et al., Nature 380:207 (1996)). Nucleic acids cancomprise other analog nucleic acids including those with positivebackbones (see, e.g., Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097(1995); non-ionic backbones (see, e.g., U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew.Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem.Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597(1994); Chapters 2 and 3, ASC Symposium Series 580, “CarbohydrateModifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook;Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffset al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743(1996)) and non-ribose backbones, (see, e.g., U.S. Pat. Nos. 5,235,033and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook). Nucleic acids can comprise one or more carbocyclicsugars (see, e.g., Jenkins et al., Chem. Soc. Rev. (1995) pp 169-176).These modifications of the ribose-phosphate backbone can facilitate theaddition of labels, or increase the stability and half-life of suchmolecules in physiological environments.

In some cases, oligonucleotides are used as probes. An “oligonucleotide”as used herein can comprise a single-stranded nucleic acid.Oligonucleotides can be from 2 to about 1000 nucleotides long.Oligonucleotides can be from 2 to about 500 nucleotides in length.Oligonucleotides can be from about 10 to about 100 nucleotides long.Oligonucleotides can be from about 20 to about 50 nucleotides in length.In methods, devices, and systems of the present disclosure, probes canbe attached to a solid substrate. Probes can be bound to a substratedirectly or via a linker. Linkers can comprise, for example, aminoacids, polypeptides, nucleotides, or oligonucleotides.

The solid substrate can be biological, non-biological, organic,inorganic, or a combination of any of these. The substrate can exist asone or more particles, strands, precipitates, gels, sheets, tubing,spheres, containers, capillaries, pads, slices, films, plates, slides,or semiconductor integrated chips, for example. The solid substrate iscan be flat or can take on alternative surface configurations. Forexample, the solid substrate can contain raised or depressed regions onwhich synthesis or deposition takes place. In some examples, the solidsubstrate can be chosen to provide appropriate light-absorbingcharacteristics. For example, the substrate can be a polymerizedLangmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO₂,SiN₄, modified silicon, the top dielectric layer of a semiconductorintegrated circuit (IC) chip, or any one of a variety of gels orpolymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride,polystyrene, polycarbonate, or combinations thereof.

The plurality of probes can be located in one or more addressableregions on a solid substrate, herein referred to as “pixels.” In somecases, a solid substrate comprises at least about 2, 3, 4, 5, 6, or7-10, 10-50, 50-100, 100-500, 500-1,000, 1,000-5,000, 5,000-10,000,10,000-50,000, 50,000-100,000, 100,000-500,000, 500,000-1,000,000 orover 1,000,000 pixels with probes. In some cases, a solid substratecomprises at most about 2, 3, 4, 5, 6, or 7-10, 10-50, 50-100, 100-500,500-1,000, 1,000-5,000, 5,000-10,000, 10,000-50,000, 50,000-100,000,100,000-500,000, 500,000-1,000,000 or over 1,000,000 pixels with probes.In some cases, a solid substrate comprises about 2, 3, 4, 5, 6, or 7-10,10-50, 50-100, 100-500, 500-1,000, 1,000-5,000, 5,000-10,000,10,000-50,000, 50,000-100,000, 100,000-500,000, 500,000-1,000,000 orover 1,000,000 pixels with probes.

In some cases it is useful to have pixels which do not contain probes.Such pixels can act as control spots in order to increase the quality ofthe measurement, for example, by using binding to the spot to estimateand correct for non-specific binding.

In some examples, it is useful to have redundant pixels which haveidentical probe sequences to another pixel but physically may not beadjacent or in proximity to the other pixel. The data acquired by suchprobe arrays may be less susceptible to fabrication non-idealities andmeasurement errors.

In some cases, labels are attached to the probes within the pixels, inaddition to the labels that are incorporated into the targets. In suchsystems, captured targets can result in two labels coming into intimateproximity with each other in the pixel. As discussed before,interactions between specific labels can create unique detectablesignals. For example, when the labels on the target and probe,respectively, are fluorescent donor and acceptor moieties that canparticipate in a fluorescent resonance energy transfer (FRET)phenomenon, FRET signal enhancement or signal quenching can be detected.

Temperature Controller

A temperature controller can establish a specific temperature for thesolution in the reaction chamber, and/or create a temperature profilethat requires heating and/or cooling. A temperature controller caninclude a feedback control system that measures the temperature, usingtemperature sensors (such as a thermistor or a thermocouple), and, basedon the measured temperature, add or remove heat from the reactionchamber using thermal devices (such as Peltier devices or resistiveheaters). Temperature controllers can comprise heat sinks for removingheat. Temperature controllers can be integrated into an array. Thetemperature of an array can be controlled by individual pixel, by arrayregions or sub-regions, or on an array-wide scale.

Temperature controllers can change the temperature of a substrate,reaction chamber, or array pixel. The rate of temperature change can beabout 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20° C./minute. The rateof temperature change can be at least about 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20° C./minute. The rate of temperature change can beat most about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20° C./minute.Temperature controllers can change temperature at a linear rate (e.g.,5° C./second). Alternatively, temperature controllers can changetemperature at a non-linear rate. Temperature controllers can increaseor decrease temperature.

Detectors

The present disclosure provides detectors that may be used to detectsignals. Such signals can be used for nucleic acid hybridizationthermodynamics, such as melt curve analysis. Such detectors can beoptical detectors for measuring optical signals, electrochemicaldetectors for measuring electrochemical signals, or electrostaticdetectors for measuring charge.

Signals detected by a detector can include signals conveying informationabout the presence, absence, and/or quantity of the labels, includingthe level of activity of labels at all pixels in real time and duringthe amplification process. Signals can be optical, such as fluorescenceor chemi-luminescence. Signals can be electrical, such aselectrochemical signals, electrostatic signals, resistance, capacitance,or inductance. Signals can be processed, including normalization to abackground signal. Signals can be detected in real-time.

Examples of optical detectors include but are not limited tocharge-coupled device (CCDs) arrays (including cooled CCDs),complementary metal-oxide-semiconductor (CMOS) imagers, n-typemetal-oxide semiconductor (NMOS), active-pixel sensors (APS), orphotomultiplier tubes (PMTs). Detectors can also includewavelength-selective components such as optical filters to allowmeasurement of selective wavelengths. Examples of other detectorsinclude electrodes.

The detector can sample (e.g., acquire measurements) at a rate of atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 90, 120, 150, 180, 210, 240, 270, 300, 400,500, 1000, 10,000 times per minute.

The detector can comprise a light source. The light source can be used,for example, to excite fluorescence and/or colorimetric labels. Thelight source can comprise at least one lamp, such as an incandescent,halogen, fluorescent, gas-discharge, arc, or light emitting diode (LED).The light source can comprise a laser. The light source can produce aspecific wavelength or range or wavelengths, such as UV. The lightsource can comprise filters for controlling the output spectrum,wavelength, or wavelengths. The light source can comprise multiple lightsources, of the same or of different types, which can be used separatelyor in combination.

The detector can comprise various optical elements, including but notlimited to filters, lenses, collimators, mirrors, reflectors, beamsplitters, and diffusers. The detector can comprise a filter or filters,including but not limited to wavelength filters (e.g., color filters, UVfilters, IR filters), dichroic filters, and polarizing filters. Thefilters can comprise multiple filters, of the same or of differenttypes, which can be used separately or in combination. The detector cancomprise elements (e.g., signal processing unit) for removing imagedistortion or aberration, such as barrel or fisheye distortion,pincushion distortion, mustache distortion, monochromatic aberrations(e.g., piston, tilt, defocus, spherical aberration, coma, astigmatism,field curvature, image distortion), or chromatic aberrations (e.g.,axial, longitudinal, lateral, transverse). Such elements can comprisecomputer systems programmed to implement instructions for partially orfully correcting image distortion. For example, Brown's distortion modelor the Brown-Conrady model can be used to correct for radial distortionand tangential distortion.

In some examples, the detector can measure emitted photons coming fromindividual pixels. These photons can be correlated to the presenceand/or activity of optical labels in that area.

In some cases, the detector comprises an integrated biosensor array,which may be built using CMOS integrated circuit (IC) fabricationprocesses (Plummer J. D. et al., “Silicon Technologies: Fundamentals,Practice, and Modeling,” Prentice Hall Electronics and VLSI Series,2000). In such systems, herein referred to as “CMOS biochips”, the probearray can be placed on top of a CMOS biochip. Examples of such systemsmay be found in, for example, U.S. Patent Pub. Nos. 2010/0122904,2013/0345065, 2014/0001341, 2014/0318958, 2014/0011710, 2012/0168306,2013/0225441, 2012/0077692, 2007/0099198, 2008/0081769, 2008/0176757 and2008/0039339, and U.S. Pat. Nos. 8,637,436, 8,048,626, and 8,518,329,each of which is entirely incorporated herein by reference.

Detection Methods

Parallel detection of nucleic acid (e.g., DNA) hybridization reactionsas a function of temperature in real time to evaluate hybridizationthermodynamics can be performed by interaction between an immobilizedprobe labeled with an energy donor (e.g., a fluorophore) at a specificpixels and a target labeled with an energy acceptor (e.g., a quencher)that is present in the reaction chamber. Detection can also be performedby interaction between an intercalator and interacting probes andtargets in a similar setting. In either case, the temperature of thereaction chamber is typically varied, while an optical detectorcontinually measures the signal in real time, to capture the amount ofhybridized targets at individual pixels and evaluate whether thehybridization reaction is favorable or not in that given temperature atthat pixel.

It is important to emphasize here that the following method all includeoptical labels, specifically fluorescent and/or quencher labels.However, signals that signify hybridization reactions are only generatedat, and are confined to the pixels of the addressable array while thereaction volume which includes all the targets creates minimumbackground optical signal. This unique characteristic not only improvethe detectable signal-to-interference (or signal-to-noise), but alsoenables multiplexing capabilities as the pixel-level measurementsremains independent of one another. This is despite the fact that thereaction chamber and aqueous sample is shared among all of them.

End-labeled Targets with Donor Probes

The probe and the target can be both end-labeled. For example, FIG. 2shows a nucleic acid target labeled with an energy acceptor label at oneend (e.g., 5′-end). Such a target can be, for example, one or moreamplicons of a PCR reaction in which the primers are labeled with anenergy acceptor label. In Method A, as shown in FIG. 2, prior tobinding, the donor fluorophore on the probe is actively radiating signalin presence of an optical excitation source with wavelengths that matchthe excitation (absorption) spectrum of that donor molecule. Once theprobe hybridizes to the target, the acceptor gets into the proximity ofthe donor and through energy transfer reduces the signal that isradiating from the donor labeled probe. In Method B, shown in FIG. 2,the hybridization of the target to the probe involves a hairpin loopforming in the target which specifically places the donor and acceptorin intimate proximity. This is done to ensure efficient interactionbetween the donor and acceptor and can be achieved by having the 3′-endof the probe sequence partially matching the 5′-end of the target, wherethe acceptor label resides. In either of these cases, the reduction indonor signal resulting when the target hybridizes to the probe can bedetected and correlated to the hybridization reaction the probe andtarget.

In some embodiments, the acceptor can be a non-radiative label, such asa quencher molecule.

Unlabeled Targets

Alternative setups can be put together in which the target is unlabeled.For example, FIG. 3 shows a nucleic acid target and a probe interactingwith an intercalator while an optical excitation source with wavelengthsspecific to the intercalator excitation (absorption) spectrum ispresent. In Method A, shown in FIG. 3, the intercalator molecules, whichare present and free roaming, in the reaction chamber, are inactive whenin the presence of the probe with unbound target; once the targethybridizes to the probe, the intercalator within the hybridized complexbecome activated and radiate a signal matching the emission spectrum ofthe intercalator indicating hybridization at that pixel. In Method B,shown in FIG. 3, the probe is labeled with an energy acceptor capable ofaccepting energy from the intercalator; once the target hybridizes theprobe, energy from the activated intercalator is harvested by the energyacceptor. If the acceptor is fluorophore, the radiated signal indicateshybridization (Howell, W M, Jobs, M, and Brooks, A J, “iFRET: animproved fluorescence system for DNA-melting analysis,” Genome Res. 2002September; 12(9):1401-7). In either case, the increased signal (fromintercalator or acceptor fluorophore, respectively), triggered by thetarget attachment to the probe is detected and correlated to thehybridization between the probe and target at specific temperatures.

Labeled Targets

The nucleic acid target can be labeled by multiple acceptors. Such atarget can be, for example, one or more amplicons of a PCR reaction inwhich acceptor-modified dNTPs are used. FIG. 4 shows a probe labeled adonor and a target with multiple energy acceptor labels. Prior to targethybridization, the probe label is radiating signal in presence of anoptical excitation source with wavelengths matching the excitation(absorption) spectrum of the donor. Once hybridization occurs, theenergy acceptors on the target can accept energy from the energy donor,effectively deactivating the donor and quenching its signal. Thereduction in energy donor signal resulting when the probe binds to thetarget can be detected and correlated to the hybridization between theprobe and target at that temperature.

In some embodiments, the acceptor can be a non-radiative label, such asa quencher molecule.

Generating Results for Parallel Melt Curve Analysis (MCA)

The detection methods described herein can be used to conduct a parallelDNA melt curve analysis (MCA). As described further in this disclosure,binding or hybridization between oligonucleotide probes and targets canresult in a change in signal in individual pixels. Such changes in thesignal can be an increase in signal or a decrease in signal, dependingon the detection method used. Conditions can be controlled and changedto alter the amount or rate of hybridization between a target and aprobe. For example, temperature can be increased to decrease the bindingbetween (i.e., “melt”) the target and the probe.

MCA can be used to detect differences in target hybridization todifferent probes. For example, nucleic acid targets can comprisedifferences in sequence (e.g., SNPs), which can affect the bindingbetween a target and a given probe. These differences can be observed asdifferences in the melt curve at different pixels or at a single pixelat different experiments. In another example, two nucleic acid targetscan differ in length, such as from an insertion or deletion (indel) orvarying number of sequence repeats. This length difference can bedetected through MCA, for example by varying length between a label anda probe binding target sequence location in the target nucleic acid.

FIG. 5 shows an example of how MCA may be performed in parallel usingmethods of the present disclosure, such as use of end-labeled targetswith donor probes. In this example, signals from three pixels are shown.Two of these pixels include probes that have matching targets (Target 1and Target 2) in the reaction chamber with dissimilar matchingsequences, while the third pixel include a probe that is designedspecifically to not to hybridize to any sequence within the sample. Thesignals generated from these pixels are, Signal 1, Signal 2, andControl, respectively. As shown in the measured signals of FIG. 5, astemperature is increased to “melt” the targets from the probe, the rawsignals show a non-monotonic increase in Signal 1 and Signal 2 yet witha different profile. The control signal, however, can decrease due, forexample, to reduction in the quantum efficiency (e.g., brightness) ofthe donor fluorophore as a function of temperature. As evident in thenormalized signal graph, once the control is used to calibrate out thetemperature dependency of the fluorophore used, the MCA signals of bothTarget 1 and Target 2 hybridization become much more apparent andclearly show Target 1 once has a more stable structure compared toTarget 2.

Integrated Detectors

Methods of the present disclosure can be implemented using integrateddetectors. An example advantage of using integrated biosensors, ratherthan conventional detection apparatuses, is the drastic reduction issize and lower cost. Furthermore, integrated biosensor arrays can bemanufactured using semiconductor integrated circuit (IC)micro-fabrication processes, e.g., complementarymetal-oxide-semiconductor (CMOS), which can offer unmatched reliability,high-volume manufacturing, and reliability. Examples of sensors that maybe used with integrated biosensors arrays of the present disclosure areprovided in U.S. Patent Pub. Nos. 2010/0122904, 2013/0345065,2014/0001341, 2014/0318958, 2014/0011710, 2012/0168306, 2013/0225441,2012/0077692, 2007/0099198, 2008/0081769, 2008/0176757 and 2008/0039339,and U.S. Pat. Nos. 8,637,436, 8,048,626, and 8,518,329, each of which isentirely incorporated herein by reference.

In such arrangements, each sensor element can be addressable and caninclude its own probe. Such sensor element may be a biosensor. The arraycan comprise a number of individual biosensors, such as at least about100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, 35000,40000, 45000, 50000, 55000, 60000, 65000, 70000, 75000, 80000, 85000,90000, 95000, or 100000 integrated biosensors. The density of individualbiosensor in the array can be at least about 100, 200, 300, 400, 500,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 biosensorpixels per mm².

A biosensor in the array can comprise a photo-sensor, such as aphotodiode. Each biosensor can also be associated with temperaturecontrol elements as well, such as heaters and temperature sensors (e.g.,thermocouples, thermistors). The biosensor array can comprise opticalfilters, such as emission filters, between the photo-sensors and thereaction chambers or array pixels as described in, for example, in U.S.Patent Pub. Nos. 2010/0122904, 2013/0345065, 2014/0001341, 2014/0318958,2014/0011710, 2012/0168306, 2013/0225441 and 2008/0081769, and U.S. Pat.Nos. 8,637,436 and 8,518,329, each of which is entirely incorporatedherein by reference.

For example, FIG. 6 shows an optical CMOS integrated biosensor detector(FIG. 6, top left) comprising a 32 by 32 array of optical biosensors(FIG. 6, top right). Each optical biosensor occupies an area of 100μm×100 μm. The optical biosensor array has a total area of 3.2 mm×3.2mm. Each biosensor comprises an integrated CMOS photodiode sensor, andan emission filter is located between the CMOS integrated sensors andthe reaction chamber of the associated array pixel (FIG. 6, bottomleft). The heat of the array can be controlled by heaters (FIG. 6,bottom right).

FIG. 7 shows example circuit architecture for an optical CMOS biochip.Each of the 1024 pixels comprises a reaction chamber associated with aphotodiode circuit, separated by an emission filter. Each pixel furthercomprises a heater, a digital controller, and signal input/output forcalibration and data collection. The biochip further comprises a digitalcontroller. The digital controller interfaces with a scan module capableof row/column selection of biochip pixels for receiving data. Thedigital controller also interfaces with a thermal controller capable ofcontrolling the on-chip temperature. A power management system providespower to the pixels and the thermal controller. Features of the opticalCMOS biochip are described in, for example, U.S. Patent Pub. Nos.2010/0122904, 2013/0345065, 2014/0001341, 2014/0318958, 2014/0011710,2012/0168306, and U.S. Pat. No. 8,518,329, each of which is entirelyincorporated herein by reference, which are entirely incorporated hereinby reference.

Computer Control Systems

The present disclosure provides computer control systems that areprogrammed to implement methods of the disclosure. FIG. 11 shows acomputer system 1101 that is programmed or otherwise configured toconduct chemical analysis, such as melt curve analysis. The computersystem 1101 can regulate various aspects of chemical analysis (e.g.,melt curve analysis) of the present disclosure, such as, for example,temperature, reagent handling, and detection. The computer system 1101can be an electronic device of a user or a computer system that isremotely located with respect to the electronic device. The electronicdevice can be a mobile electronic device.

The computer system 1101 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 1105, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 1101 also includes memory or memorylocation 1110 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 1115 (e.g., hard disk), communicationinterface 1120 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 1125, such as cache, othermemory, data storage and/or electronic display adapters. The memory1110, storage unit 1115, interface 1120 and peripheral devices 1125 arein communication with the CPU 1105 through a communication bus (solidlines), such as a motherboard. The storage unit 1115 can be a datastorage unit (or data repository) for storing data. The computer system1101 can be operatively coupled to a computer network (“network”) 1130with the aid of the communication interface 1120. The network 1130 canbe the Internet, an internet and/or extranet, or an intranet and/orextranet that is in communication with the Internet. The network 1130 insome cases is a telecommunication and/or data network. The network 1130can include one or more computer servers, which can enable distributedcomputing, such as cloud computing. The network 1130, in some cases withthe aid of the computer system 1101, can implement a peer-to-peernetwork, which may enable devices coupled to the computer system 1101 tobehave as a client or a server.

The CPU 1105 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 1110. The instructionscan be directed to the CPU 1105, which can subsequently program orotherwise configure the CPU 1105 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 1105 can includefetch, decode, execute, and writeback.

The CPU 1105 can be part of a circuit, such as an integrated circuit.One or more other components of the system 1101 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 1115 can store files, such as drivers, libraries andsaved programs. The storage unit 1115 can store user data, e.g., userpreferences and user programs. The computer system 1101 in some casescan include one or more additional data storage units that are externalto the computer system 1101, such as located on a remote server that isin communication with the computer system 1101 through an intranet orthe Internet.

The computer system 1101 can communicate with one or more remotecomputer systems through the network 1130. For instance, the computersystem 1101 can communicate with a remote computer system of a user(e.g., a lab technician). Examples of remote computer systems includepersonal computers (e.g., portable PC), slate or tablet PC's (e.g.,Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g.,Apple® iPhone, Android-enabled device, Blackberry®), or personal digitalassistants. The user can access the computer system 1101 via the network1130.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 1101, such as, for example, on thememory 1110 or electronic storage unit 1115. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 1105. In some cases, thecode can be retrieved from the storage unit 1115 and stored on thememory 1110 for ready access by the processor 1105. In some situations,the electronic storage unit 1115 can be precluded, andmachine-executable instructions are stored on memory 1110.

The code can be pre-compiled and configured for use with a machine havea processor adapted to execute the code, or can be compiled duringruntime. The code can be supplied in a programming language that can beselected to enable the code to execute in a pre-compiled or as-compiledfashion.

Aspects of the systems and methods provided herein, such as the computersystem 1101, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such memory (e.g., read-only memory, random-access memory,flash memory) or a hard disk. “Storage” type media can include any orall of the tangible memory of the computers, processors or the like, orassociated modules thereof, such as various semiconductor memories, tapedrives, disk drives and the like, which may provide non-transitorystorage at any time for the software programming. All or portions of thesoftware may at times be communicated through the Internet or variousother telecommunication networks. Such communications, for example, mayenable loading of the software from one computer or processor intoanother, for example, from a management server or host computer into thecomputer platform of an application server. Thus, another type of mediathat may bear the software elements includes optical, electrical andelectromagnetic waves, such as used across physical interfaces betweenlocal devices, through wired and optical landline networks and overvarious air-links. The physical elements that carry such waves, such aswired or wireless links, optical links or the like, also may beconsidered as media bearing the software. As used herein, unlessrestricted to non-transitory, tangible “storage” media, terms such ascomputer or machine “readable medium” refer to any medium thatparticipates in providing instructions to a processor for execution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 1101 can include or be in communication with anelectronic display 1135 that comprises a user interface (UI) 1140 forproviding, for example, temperature values, temperature control,detector data, and fluid handling. Examples of UI's include, withoutlimitation, a graphical user interface (GUI) and web-based userinterface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 1105. Thealgorithm can, for example, control the temperature of array pixels andcollect and process data.

EXAMPLES Example 1 Melt Curve Analysis (MCA) on the Integrated BiosensorArray

An array of probes is contacted with first targets and second targets,and binding occurs between the probes and the targets (see, e.g., FIG.8). The temperature is increased over time using the on-chip heaters, asshown in FIG. 8B (upper graph), including time points of T=40° C. at t=0minutes, T=48° C. at t=10 minutes, T=57° C. at t=15 minutes, T=67° C. att=20 minutes, T=76° C. at t=25 minutes, and T=85° C. at t=30 minutes(FIG. 8A). The integrated biosensor array blocks the excitation signaland collect the emission signals from the array, and melt curves areproduced for the first targets (Signal (1)) and the second targets(Signal (2)) with normalized signal compared to temperature (FIG. 8B,lower graph). Probe and target sequences used are shown in FIG. 8C.

The donor label in this experiment is HEX and the acceptor (quencher) isIowa Black.

Example 2 Intercalator-Based Melt Curve Analysis (MCA) on the IntegratedBiosensor Array

An array of probes is contacted with first targets and second targets,and binding occurs between the probes and the targets in the presence ofSYBR Green intercalator (see, e.g., FIG. 9, right-hand side). In theabsence of binding between the probe and the target, the intercalator isnot active; when the probe and target bind, the intercalator activatesand radiates signal. The temperature is increased using on-chip heatersover time. The integrated biosensor array blocks the excitation signaland collect the emission collect emission signals from the array, andmelt curves are produced for the first targets (Signal (1)) and thesecond targets (Signal (2)) with normalized signal compared totemperature (FIG. 9, graph). Probe and target sequences used are shownin FIG. 9, lower portion.

Example 3 Fluorophore-Quencher-Based Melt Curve Analysis (MCA) on theIntegrated Biosensor Array

An array of first, second, third, and fourth probes is contacted withfirst, second, third, fourth targets, and binding occurs between theprobes and the targets (see, e.g., FIG. 10A, right-hand side). Theprobes are end-labeled with energy donors, and the targets areend-labeled with energy acceptors. The temperature is increased overtime. The biochip sensors collect signal from the array, and melt curvesare produced for the first targets (Signal 1), the second targets(Signal 2), the third targets (Signal 3), and the fourth targets (Signal4) with normalized signal compared to temperature (FIG. 10A, graph).Probe and target sequences used are shown in FIG. 10B.

The donor label in this experiment is HEX and the acceptor (quencher) isIowa Black.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A method for assaying a presence of a targetnucleic acid molecule in a sample, comprising: (a) providing a chipcomprising a sensor that is adjacent to a sample chamber, wherein saidsample chamber is configured to retain said sample having or suspectedof having said target nucleic acid molecule, wherein said sensorincludes (i) a first surface including a probe that selectively couplesto said target nucleic acid molecule, (ii) an emission layer below saidsurface, (iii) an optical detector below said emission layer, whereinsaid optical detector detects at least one signal from said sample upontransmission through said emission layer, which at least one signal isan optical signal generated upon interaction between said probe and saidtarget nucleic acid molecule, and (iv) a second surface including acontrol probe that does not selectively couple to said target nucleicacid molecule, wherein said control probe provides a control signalseparate from said optical signal; (b) providing said sample in saidsample chamber under conditions that permit said probe to selectivelycouple to said target nucleic acid molecule; (c) subjecting said firstsurface and second surface to a temperature change while said sample isin said sample chamber; (d) using said sensor to measure said at leastone signal in real-time while subjecting said first surface and secondsurface to said temperature change; wherein said at least one signal isnot measured during amplification of said target nucleic acid molecule,wherein said at least one signal is generated upon energy transferbetween an energy acceptor coupled to said target nucleic acid moleculeand an energy donor coupled to said probe, and (e) generating signalversus temperature data using measurements of said at least one signalwith said temperature change, wherein said signal versus temperaturedata is normalized against measurements of said control signal whilesaid second surface is subjected to said temperature change.
 2. Themethod of claim 1, wherein said probe is an oligonucleotide.
 3. Themethod of claim 2, wherein said sample is provided in said samplechamber under conditions that permit said oligonucleotide to hybridizeto said target nucleic acid molecule.
 4. The method of claim 3, whereina sequence of said target nucleic acid molecule forms a hairpin loopstructure when hybridized to said oligonucleotide.
 5. The method ofclaim 1, wherein said sensor is in an array of a plurality of sensors insaid chip.
 6. The method of claim 1, wherein said optical signal isindicative of an interaction between said energy acceptor and saidenergy donor pair.
 7. The method of claim 6, wherein said energyacceptor quenches optical activity of said energy donor.
 8. The methodof claim 1, wherein said optical signal is indicative of an activity ofan optically-active species that is a fluorophore.
 9. The method ofclaim 1, wherein said detecting comprises measuring an increase in saidat least one signal relative to background.
 10. The method of claim 1,further comprising, prior to (a), (i) providing a reaction mixtureincluding a biological sample having or suspected of having a templatenucleic acid molecule as a precursor of said target nucleic acidmolecule, at least one primer that is complementary to said templatenucleic acid molecule, and a polymerase, and (ii) subjecting saidreaction mixture to a nucleic acid amplification reaction underconditions that yield said target nucleic acid molecule in said sample.11. The method of claim 10, wherein said at least one primer has asequence that is selective for a single nucleotide polymorphism (SNP) ina sequence of said target nucleic acid molecule.
 12. The method of claim10, wherein said nucleic acid amplification is asymmetric nucleic acidamplification.
 13. The method of claim 1, wherein said chip iselectrically coupled to a computer processor that receives an electricalsignal corresponding to said at least one signal from said sensor anddetermines said presence or absence of said target nucleic acid moleculefrom said electrical signal.
 14. The method of claim 1, wherein, in (c),said first and second surfaces are subjected to said temperature changeat an average rate from about 1° C./min to 20° C./min.
 15. The method ofclaim 1, wherein said probe is coupled to said first surface via alinker.
 16. The method of claim 1, wherein, when said at least onesignal is indicative of said presence of said target nucleic acidmolecule, said target nucleic acid molecule is detected at a sensitivityof at least about 90%.
 17. The method of claim 1, further comprisingdetermining a single nucleotide polymorphism (SNP) in a sequence of saidtarget nucleic acid molecule using said signal versus temperature data.18. The method of claim 1, further comprising (i) performingmeasurements of said control signal from an additional sensor, and (ii)normalizing said signal versus temperature data against measurements ofsaid at least one control signal.
 19. The method of claim 1, whereinsaid sensor comprises a plurality of optical detectors.
 20. The methodof claim 1, wherein said first surface and said second surface are thesame surface.
 21. The method of claim 1, wherein said sensor furthercomprises an additional optical detector that detects said controlsignal.
 22. The method of claim 1, further comprising a third surfaceincluding an additional probe that selectively couples to an additionaltarget nucleic acid molecule in said sample.
 23. The method of claim 1,wherein an energy acceptor is coupled to one or more nucleotides of saidtarget nucleic acid molecule, and wherein an energy donor is coupled tosaid probe.